The thiazide-sensitive sodium chloride cotransporter (NCC) is one of the key determinants of salt balance and thus systemic blood pressure. However, in contrast to other important effectors of salt balance we know little about how hormonal and physiological signals alter activity of this cotransporter. Preliminary data demonstrates the role of the ERK1/2 MAPK (Extra-cellular signal-Regulated Kinases 1 and 2 Mitogen-Activated Protein Kinases) pathway in the ubiquitination and endocytosis of NCC. With-No-Lysine Kinase 1 and 4 (WNK1 and WNK4), Ste20-related proline alanine-rich kinase (SPAK) and Oxidative Stress Response-1 (OSR1) are also important regulators of NCC, and there is data that some hormones regulate NCC. However, a link between hormones and the intracellular pathways that mediate the effects of hormones on NCC has not been established. The broad hypothesis of the proposed investigation is that NCC is subject to hormonal regulation that proceeds through modulation of the kinases ERK1/2, SPAK/OSR and WNKs. Aim 1 will define the hormonal regulation of NCC through activation of ERK1/2 MAPK. ERK1/2 MAPK will be examined as a central mediator of the hormonal/physiological effects of Epidermal Growth Factor (EGF) and the Calcium-sensing Receptor (CaSR) on NCC. The mechanism of ERK1/2 activation and the roles of ubiquitination and endocytosis in this process will be thoroughly examined for each hormone, tracing a pathway from hormonal stimulus to functional effect. Aim 2 will determine the hormonal regulation of NCC by Aldosterone through ERK1/2 MAPK and SPAK/OSR and the mechanisms underlying these effects. ERK1/2 inhibition and SPAK/OSR activation by Aldosterone may lead to increased NCC activity. The role of WNKs in these pathways will be thoroughly examined. Aim 3 will determine the underlying molecular mechanisms mediating hormonal regulation of NCC by Angiotensin II. The roles of each of these kinase pathways will be examined with a particular emphasis on WNKs. Mammalian cell culture, isolated tubule microperfusion and whole animal experimentation will be utilized to examine effects at the cellular/molecular, tubular and organismal level. Knowledge of hormonal regulation of blood pressure has played a critical role in the development of new therapies to treat hypertension, the single risk factor that contributes the most to worldwide mortality. These studies will provide vital information on the regulation of this important effector of blood pressure homeostasis. PUBLIC HEALTH RELEVANCE: The sodium chloride cotransporter (NCC) is an important salt absorptive pathway in the mammalian kidney that is the site of action of one of the most effective classes of anti- hypertensive medications and plays a key role in genetic disorders of hypertension. Despite the importance of this cotransporter in human disease, we know little about its hormonal regulation. This investigation of the hormonal regulation of this key effector of blood pressure will provide invaluable insight into the pathogenesis of Hypertension.